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Sample GSM1836785 Query DataSets for GSM1836785
Status Public on May 10, 2016
Title PHH_WO_LD_exp3 [miRNA]
Sample type RNA
Source name PHH_WO_LD_exp3
Organism Homo sapiens
Characteristics cell type: primary human hepatocytes
main source: liver
study type: Wash out effect
assay type: ex vivo
strain: Hu4227, Hu8150, Hu4197
agent: aflatoxin B1
dose: 0.3
dose unit: uM
dose duration: 5
duration unit: days
dose frequency: daily
vehicle: DMSO
route: medium
Treatment protocol After quick thawing in a water bath at 37°C, viability of the cells was checked by a Trypan blue (CAS no. 72-57-1, Sigma–Aldrich) exclusion test as instructed in the supplier’s protocol (Invitrogen, 2012). All viability scores after thawing were in agreement with those listed by the supplier. Before AFB1 treatment, cells were allowed to acclimatize for 48 hours. This is needed for the hepatocytes to restore an in vivo like cellular configuration and enzyme expression levels as optimally as possible.AFB1 doses causing minimal (IC20) cytotoxicity after 5 days of repetitive daily exposure were established by means of the MTT assay (23). Furthermore, crucial liver function enzymes such as lactate dehydrogenase (LDH) and alanine transaminase (ALT) were measured. LDH and ALT were spectrophotometrically determined on a Cobas 8000 Modular Analyser (Roche Diagnostics, Basel, Switzerland). Based on this data, 0.3 µM of AFB1, was selected for the main experiment.
Growth protocol Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well and 6-well plates (700,000 cells/ml) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen, 2012). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation.
Extracted molecule total RNA
Extraction protocol Cells were cultured in triplicate in 24 wells (RNA) or 6 wells (DNA) in a collagen sandwich layer. Following 5 days of repetitive daily exposure the treatment was removed for 3 days (washout period), then cells lysates were harvested for DNA and total RNA isolation. At the end of washout, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment. Upon purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 8, were used. Samples were stored at -80ºC until RNA hybridization.
Label Cy3
Label protocol MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays.
Hybridization protocol The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
Scan protocol Samples were labeled according to the manufacturers protocol.
Description Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays
Data processing The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 ( for quality control with an in-house developed pipeline. Filtering and normalization was performed using AgiMicroRna (32). Total gene signals were log2-transformed and quantile-normalized. The selection criteria for determining differentially expressed microRNAs (DE-miRs) were identical to those for DEGs.
Submission date Jul 30, 2015
Last update date May 10, 2016
Contact name Linda Rieswijk
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 50
City Maastricht
ZIP/Postal code 6229 ER
Country Netherlands
Platform ID GPL18402
Series (2)
GSE71545 Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma [miRNA]
GSE71549 Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma

Data table header descriptions
VALUE Quantile normalized intensity signals

Data table
miRNABrightCorner30 10.95978464
Blank -1
hsa-miR-1272 -1
hsa-miR-215 5.859716574
hsa-miR-3651 6.188390704
hsa-miR-642b-5p -1
hsa-miR-4535 -1
hsa-miR-616-3p -1
hsa-miR-519e-3p -1
hsa-miR-499b-3p -1
hsa-miR-645 -1
hsa-miR-520e 2.889129864
hsa-miR-3687 -1
hsa-miR-4439 -1
hsa-miR-6128 -1
hsa-miR-711 -1
hsa-miR-3936 -1
hsa-miR-4425 -1
hsa-miR-4519 -1
hsa-miR-4448 -1

Total number of rows: 2027

Table truncated, full table size 36 Kbytes.

Supplementary file Size Download File type/resource
GSM1836785_US10063773_254606410411_S01_miRNA_107_Sep09_1_1.txt.gz 6.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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